1
DEPARTMENT OF HEALTH AND HUMAN SERVICES
FOOD AND DRUG ADMINISTRATION
CENTER FOR FOOD SAFETY AND APPLIED NUTRITION
FOOD ADVISORY COMMITTEE MEETING
Advice on CFSAN'S Draft Report:
Approaches to Establish Thresholds for Major Food Allergens and for Gluten in Food
Thursday, July 14, 2005
8:30 A.M. to 5:20 P.M.
Greenbelt Marriott
6400 Ivy Lane
Grand Ballroom
Greenbelt, Maryland 20770
2
P A R T I C I P A N T S
FOOD ADVISORY COMMITTEE STANDING MEMBERS:
Richard A. Durst, Ph.D. - Acting Chairman
Jeffrey A. Barach, Ph.D. (Industry Representative)
Patrick S. Callery, Ph.D.
Dennis Gonsalves, Ph.D., M.S.
Jean M. Halloran (Consumer Representative)
Douglas C. Heimburger, M.D., M.S.
Margaret C. McBride, M.D.
Mark Nelson, Ph.D. (Industry Representative)
Carol I. Waslien Ghazaii, Ph.D., R.D.
TEMPORARY VOTING MEMBERS:
Petr Bocek, M.D., Ph.D. (Absent 7/14/05 Session)
Margaret Briley, Ph.D., R.D.
Erica Brittain, Ph.D.
Ciaran P. Kelly, M.D.
Soheila June Maleki, Ph.D.
David O. Oryang
Marc D. Silverstein, M.D.
Suzanne Teuber, M.D.
FOOD AND DRUG ADMINISTRATION:
Robert E. Brackett, Ph.D. - Director
Food and Drug Administration, CFSAN
Catherine Copp, J.D. - Senior Policy Advisor
Food and Drug Administration, CFSAN
Steven M. Gendel, Ph.D. - Senior Scientist
Food and Drug Administration
National Center for Food Safety and Technology
Rhonda Kane, M.S., R.D. - Consumer Officer
Food and Drug Administration, CFSAN
Marcia Moore, Food Advisory Committee, Executive Secretary
3
P A R T I C I P A N T S (Continued)
FOOD AND DRUG ADMINISTRATION STAFF:
Michael M. Landa, J.D. - Deputy Director for Regulatory Affairs
Food and Drug Administration, CFSAN
Stafano Luccioli, M.D. - Senior Medical Advisor
Food and Drug Administration, CFSAN
GUEST SPEAKERS:
Pekka Collin, M.D., M.P.H. - Professor
University of Tampere, Medical School, Finland
Catherine L. Copp, J.D.
Policy Advisor, CFSAN, FDA
Alessio Fasano, M.D. - Professor of Pediatrics
Medicine & Physiology and Director, the Mucosal
Biology Research Center, Center for Celiac
Research, University of Maryland School of
Medicine
Steven M. Gendel, Ph.D. - Senior Scientist
National Center for Food Safety and Technology, FDA
Rhonda R. Kane, M.S., R.D. - Consumer Safety
Officer CFSAN, FDA
Donald Kasarda, Ph.D. - Consultant and Retired
Senior Scientist, Agriculture Research Service,
USDA
Cynthia Kupper, R.D., C.D. - Executive Director
Gluten Intolerance Group of North America
Joseph A. Murray, M.D. - Professor of Medicine
The Mayo
Clinic of Rochester, Minnesota
4
C O N T E N T S
PAGE
Call to Order and Welcome and Introductions
Richard Durst, Ph.D., Acting Chairman 6
Use of Gluten Thresholds
Catherine L. Copp, J.D., CFSAN, FDA 9
Introduction to Celiac Disease
Joseph Murray, M.D. 11
Patient Perspectives on Celiac Disease
Cynthia Kupper, R.D., C.D. 69
Grains
Donald Kasarda, Ph.D., USDA 84
Question and Answer Session 108
Prospective Studies
Alessio Fasano, M.D. 114
Question and Answer Session 146
Retrospective Studies
Pekka Collin, M.D., M.P.H. 161
Question and Answer Session 182
International Perspectives on Gluten-Free
Rhonda S. Kane, M.S., R.D., CFSAN, FDA 186
Question and Answer Session 198
Public Comments:
Elaine Monarch 212
Alice Bast 220
Mary Schluckabeer 225
Tom P. Sullivan 232
Steve Taylor 240
5
C O N T E N T S
PAGE
Overview of Approaches to Establishing
Thresholds: Gluten
Steven M. Gendel, Ph.D. 253
Question and Answer Session 257
Committee Discussion:
Panel Discussion with Guest Speakers 259
Gluten and Celiac Disease - FDA Questions 287
Revisit Food Allergens 354
Adjournment
Richard Durst, Ph.D., Acting Chairman 363
6
P R O C E E D I N G S
CALL TO ORDER AND WELCOME AND INTRODUCTIONS
CHAIRMAN DURST: Good morning. I would
like to call the meeting to order. All right, I
would like to welcome everyone back and also
welcome new participants in our meeting this
morning.
For those of you who weren't here
yesterday, there is a "Conflict of Interest
Statement" over on the table, if you want to refer
to that at all, otherwise I would also ask again
that maybe our participants or our members of the
Food Advisory Committee would introduce themselves
again for the benefit of those who were not here
yesterday.
I am Dick Durst, professor [of]chemistry at
Food Science and Technology Department at Cornell
University.
Marc, would you start it off?
DR. SILVERSTEIN: Marc Silverstein, I'm a
general internist and geriatrician at Baylor Health
Care System
in Dallas.
7
DR. TEUBER: Suzanne Teuber, I am an
allergist at UC-Davis.
MR. ORYANG: I am David Oryang. I am a
risk analyst and agricultural engineer at the
United States Department of Agriculture, Animal and
Plant Health Inspection Service.
DR. KELLY: Good morning. Ciaran Kelly, I
am a gastroenterologist at Harvard Medical School
in Boston.
DR. MALEKI: I am Soheila Maleki. I am a
scientist with the USDA.
DR. BRITTAIN: Erica Brittain, I am a
statistician at the National Institute of Allergy
and Infectious Disease.
DR. BRILEY: Margaret Briley, University
of Texas at Austin, nutritionist.
MRS. MOORE: Marcia Moore, I am the
executive secretary for the Food Advisory Committee
and the Food and Drug Administration.
DR. WASLIEN: I am Carol Waslien,
nutritional epidemiologist at the School of
Medicine,
the University of Hawaii.
8
DR. BRILEY: I am Margaret McBride, child
neurologist at Akron Children's Hospital.
DR. CALLERY: Pat Callery, West Virginia
University, pharmaceutical scientist.
DR. GONSALVES: I am Dennis Gonsalves, a
scientist at USDA.
DR. HEIMBURGER: I am Doug Heimburger, a
physician and nutrition specialist at the
University of Alabama at Birmingham.
DR. BARACH: Jeff Barach with Food
Products Association here, in Washington, D.C., in
regulatory affairs.
DR. NELSON: Mark Nelson with the Grocery
Manufacturers Association here, in Washington,
D.C., and I am responsible for scientific and
regulatory policy.
MS. HALLORAN: Jean Halloran with
Consumers Union located in Yonkers, New York, and I
am director of food policy initiatives.
CHAIRMAN DURST: Thank you very much.
I would also like to remind everyone and
also for
our new people here at the meeting that
9
the "Charge" of the Committee is written out on the
meeting document. The most important thing is that
we are focusing on the "Threshold Working Group
Draft Report on Approaches to Establish Thresholds
for Major Food Allergens and for Gluten in Food."
We are not here to set any kind of
thresholds or discuss the labeling of these foods
for allergens, but strictly to make comments on the
best approaches to use for setting these
thresholds.
Did I cover everything that we need to?
(No verbal response.)
CHAIRMAN DURST: In that case, let's begin
with our first presentation. This is
Catherine Copp, the policy advisor for CFSAN, FDA,
on the use of gluten thresholds.
USE OF GLUTEN THRESHOLDS
MS. COPP: I have been asked this morning
to proceed with discussion on gluten and threshold
levels for gluten or possible thresholds for gluten
framework. It is similar to yesterday. I simply
want to
provide you with some context for the
10
evaluating the Draft Report portion that addresses
gluten in food. This is the hazard of being first.
(Slide.)
MS. COPP: Yesterday, I mentioned the Food
Allergen Labeling and Consumer Protection Act.
This is a new law that Congress passed last August.
Although it focuses primarily on allergens, food
allergens, Congress also directed FDA to address
the separate problem of gluten in food.
When I say directed, I mean that Congress
has mandated that the Agency consider and then
establish regulations according to a schedule to
define "gluten-free" for use on food labels and
also to identify the appropriate use of the term.
As with Allergens, for consumers with
celiac disease, strict avoidance of gluten at
levels that will elicit an adverse effect is the
only means to prevent potentially serious
reactions.
Accurate, complete and informative
labeling on foods can help these consumers achieve
their
goal. We believe that understanding
11
thresholds for gluten and having a sound scientific
framework for evaluating the existence of such
thresholds will help FDA develop a definition of
gluten-free and identify appropriate use of the
term. That's it.
Thank you.
CHAIRMAN DURST: Does anyone have any
comments or question on Catherine's presentation?
(No verbal response.)
CHAIRMAN DURST: Okay. We will move on
then to the presentation from Dr. Joseph Murray,
professor of medicine at the Mayo Clinic of
Rochester, Minnesota, on the introduction to celiac
disease.
INTRODUCTION TO CELIAC DISEASE
DR. MURRAY: Good morning, Committee
Members. I will be providing a general overview to
celiac disease.
(Slide.)
DR. MURRAY: First of all, we will discuss
what is celiac disease. We will discuss, briefly,
the
pathogenesis of the disease; who gets it; what
12
the treatment is, at least in a very relatively
superficial fashion. We will discuss some of the
complications and compliance issues of celiac
disease and a prognosis or future of celiac
disease.
(Slide.)
DR. MURRAY: Obviously, yesterday was
focused on food allergies. "Celiac disease" is one
of the food intolerances that is immune-mediated,
though it is not thought to be IgE-mediated; so, it
comes into the non-IgE-mediated food intolerances
that are mediated by an immune response.
(Slide.)
DR. MURRAY: Where does it happen? It
happens within the smaller intestine, predominantly
the proximal, smaller intestine is the workhorse of
the digestive system. It is this surface of the
intestinal lining that is maximally expanded by the
development of circular folds and on top of these
circular folds the so-called "villi," these villi,
shown here in a histological picture, which
maximize
the digestive surface area.
13
It is on the surface entrocytes of these
villi that most of the enzymes and in the layer
immediately above that in the lumen where most
digestion of the macromolecules from nutrition are
broken down and then absorbed. This is just a
picture. It looks like one of those shag-ply
carpets from the 1970s. This is a normal
appearance.
(Slide.)
DR. MURRAY: However, celiac disease is an
inflammatory state of the small intestinal mucosa.
It occurs in those who are genetically predisposed,
and it resolves, the damage resolves, with
exclusion of dietary gluten.
Here, on the left, is a normal intestine
with a normal villus structure; and on the right,
fully evolved celiac disease with complete
destruction.
The villi are gone, not only are they gone
but this entire intestinal mucosa is greatly
thickened and filled with inflammatory cells. This
is where
the primary site of injury occurs in
14
celiac disease.
I didn't mention it, but it is a permanent
condition. While it will heal most of the time
with exclusion of gluten, the intolerance to gluten
is permanent and will recur when the individual is
reexposed to gluten.
(Slide.)
DR. MURRAY: Now, what causes celiac
disease?
(Slide.)
DR. MURRAY: We know there are two major
components to this disease: the first is the
genetic background of predisposition.
Much of that predisposition revolves in
the HLA type, which is part of our human leucocyte
antigen-recognition system. It is how we determine
self- and non-self and generate an immune response
as appropriate and its interaction with
environmental factors, primarily the environmental
factor of gluten.
These two conspire together to produce an
immune
response that becomes out of control
15
resulting in inflammation, which we just showed to
you, that occurs primarily in the proximal small
intestine and then subsequently the consequences of
this inflammation leading to malabsorption and
symptoms.
(Slide.)
DR. MURRAY: What do we know about the
genetics of the disease? For many years, we know
there is a strong, familial predisposition to the
disease.
If you are unlucky enough to be a
monozygous twin of somebody affected with celiac
disease, your concordance rate is 80 percent. It
is not 100 percent, but it is about 80 percent. If
you are a sibling of a celiac, your chance of
having it is 10 percent. If you are a child of,
about 5 to 10 percent.
There is a very strong association with
certain HLA molecules. These are Class II MHC
molecules but particularly two types. First, DQ2
is the predominant type that is required for celiac
disease,
and in some populations DQ2 is also an
16
enabling type.
These genes, however, while they are
essentially required for the disease, are not
sufficient alone to the development of the disease.
Probably 30 to 40 percent of the Caucasian
population carry one or both of these molecules,
but most of them don't get celiac disease. There
are other HLA genes that are likely involved,
though they have not been well elucidated and
certainly not confirmed in many populations.
There are other chromosomal disorders --
Down's syndrome, Turner's syndrome, and Williams
syndrome -- that are associated with a greatly
increased risk of developing celiac disease for
reasons that are not entirely clear, but probably
are associated with the increase risk of disease in
those chromosomal disorders.
(Slide.)
DR. MURRAY: Looking at the primary
environmental trigger for the disease -- that is,
gluten -- it is basically the storage proteins that
come from
these particular cultivated grasses:
17
wheat, barley, rye, and other similar grains from
within those families. Other grasses -- for
example, rice, items such as corn, sorghum, millet,
and probably not even oats -- are not involved in
triggering the disease.
(Slide.)
DR. MURRAY: It is the storage proteins
from the endosperm compartment of the wheat kernel
particularly, and those are gliadins oar glutenins
that are thought to contain the antigenic moieties
that trigger the disease.
(Slide.)
DR. MURRAY: What is it about these wheat
proteins? Well, if you take wheat, as an example
of the others, these storage proteins are uniquely
high in certain amino acids, especially glutamines
and prolines.
Over 30 percent of the amino acids in
gliadin are glutamines. The glutamines, of course,
can be cross-linked to give the grain its
resiliency. Really the cooking ability, the
ability to
use wheat as such an effective way of
18
making things that stick together like bread, for
example, and maintain their shape, is largely a
property of these particular combinations of amino
acids.
The proline sequences that contain or
proline residues contained within the wheat
proteins also appear to form helices, and these
helices are resistant to digestion within the
mammalian gut.
(Slide.)
DR. MURRAY: This may be a key factor in
what results in the likelihood of these peptides
basically being maintained and becoming, then,
still available for the immune system to recognize
in a patient with celiac disease.
Now, gliadin molecules are presented by
these HLA types to T-cells in the intestine, and
T-cells that are specifically primed to respond to
gluten. There are certain gliadin molecules that
have a higher affinity than others for these
Class II molecules and then the T-cell receptor.
These peptides may be processed or
altered
19
within the gut, perhaps, to make them more
antigenic. They may not start out very antigenic,
but then they undergo some change within the gut
that may make them more antigenic.
It turns out that some of these peptides
that are particular immunodominant, these are the
ones that are most likely to produce an immune
response, that those immunodominant peptides may be
digestion resistant because they contain those
proline sequences that perform helix, making them
relatively poorly digestible by peptidases within
the gut.
(Slide.)
DR. MURRAY: Now, it turns out that there
is a contribution to this antigenic nature from
within the intestine itself, and this may well be
because of this enzyme tissue transglutaminase.
This is an enzyme that is present within
the gut mucosa. It is released by cells,
especially fibroblasts when they become inflamed,
and it cross-links cystine residues.
It turns out that it will also act on
20
gliadin by deamidating some of those glutamine
residues, some specific glutamine residues, to
glutamates, making it more antigenic by deamidating
that gliadin peptide and making it fit more
perfectly or with a tighter affinity into the
binding groove of the DQ2-HLA molecule, and, hence,
producing a more vigorous immune response within
the gut.
(Slide.)
DR. MURRAY: This is a schema, a
relatively simplified schema, of what I have just
talked about. We start with wheat. You look at
particularly the ethanol-soluble fraction, and
gliadin is probably broken up into smaller
peptides, but still of a sufficient size to produce
an immune response.
It is taken up across the epithelium
presented by antigen presenting cells to the
T-cells. These are T-cells that will specifically
respond to gluten then producing two types of
responses: a cellular response, characterized by
lymphocytes
producing interferon gamma and possibly
21
other cytokines. It is probably the cellular
response that leads to the "inflammatory cascade"
that produces the damaged epithelium characteristic
of celiac disease.
It also produces help to the B-cell side,
to produce plasma cells that produce antibodies.
These antibodies are directed both against the
exogenous antigen gliadin and also antibodies
against tissue transglutaminase or what was known
as an the endomysial antibody. It is not known
what the actual pathogenic role of this is, but it
is a very useful serologic or blood marker for the
disease.
I mentioned about the antigen getting
changed by tissue transglutaminase. This is a
little cartoon which shows the peptide derived from
gluten. If you change one specific glutamine to a
glutamic acid, which could be done by tissue
transglutaminase, this then binds much more
tightly. This is the HLA molecule here on the
surface of the antigen presenting cell, and it fits
more
perfectly into the T-cell receptor, producing
22
a more potent T-cell response.
(Slide.)
DR. MURRAY: Now, there are other things
that happen in the setting of celiac disease, and I
am really touching just on the surface of many of
these, but there are other things that result in
this inflammation that damage the lining of the
intestine.
For example, there are metallic proteases
that damage the structural elements that maintain
the structure that maintain villus structure.
There is endothelial injury that occurs affecting
the blood vessels in the villus. There are
antibodies, autoantibodies, that are produced that
affect actin that are involved in the site
maintaining the structure of the entrocyte itself.
(Slide.)
DR. MURRAY: Recently, there has been work
suggesting that there is a molecule called
"zonulin" that may be released in the setting of
celiac disease.
This is important because it opens up
23
tight junctions between entrocytes which may allow
even more ready access of the antigen, the foreign
antigens, between the cells into lamina propria
where antigen-presenting cells can then present
those peptides to the gluten-responsive T-cells,
further accelerating the disease.
(Slide.)
DR. MURRAY: Now, I pointed out that many
people, 30 percent or more of the Caucasian
population, carry DQ2 or DQ8. Virtually, the
entire population are exposed to gluten, but most
people don't get the disease.
There must be triggers that produce the
disease. There is evidence that suggests that
gluten in the infant diet, specifically the age of
introduction of gluten into the infant's diet, may
be important in triggering or at least producing
autoantibody markers suggestive of celiac disease
early in life.
It is not clear, however, if that changes,
whether you delay introduction or not whether that
changes,
the lifetime risk of celiac disease, but
24
it certainly seems to be important in triggering or
producing evidence in childhood at least of celiac
disease immune markers.
The amount of gluten in the child's diet
may be important. There are other events such as
pregnancy, infection, or surgeries that may bring
previously asymptomatic celiac disease to clinical
presentation.
(Slide.)
DR. MURRAY: One could speculate, and I
think this is based on some data, putting data
together, that one's risk for celiac disease starts
with your HLA type. Only those who carry HLA types
are at risk. You, then, are exposed to gluten.
Perhaps the timing of exposure is important,
developing in some individuals a sensitivity to
gluten.
Then, with the interaction of other
factors such as other genes other than HLA, other
things that may predispose one to autoimmunity
including gender and other events that may occur --
gastroenteritis, aging, postsurgical or postpartum
25
changes in the immune system that may occur -- may
lead to a loss of tolerance, inflammation, and
subsequent malabsorption.
(Slide.
DR. MURRAY: Don Kasarda, who is here once
used the term or suggested that celiac disease was
a collision between our evolution of our immune
system and our ability to recognize self and
non-self through the HLA system and our cultivation
of wheat and these other grasses. This collision
occurs in the intestine.
(Slide.)
DR. MURRAY: Now, when this collision
occurs and results in damage, how does it present?
And who gets the disease?
(Slide.)
DR. MURRAY: Well, this is classic celiac
disease, and this is the way that I certainly
learned about celiac disease. A severe
malnourished child with evidence of malnutrition
often associated with the large, swollen abdomen
but great
muscle, terrific muscle, wasting and
26
protein-calorie malnutrition with symptoms that
would occur sometime after the onset of gluten
introduction into the diet, sometime between the
age of six months and seven years of age: with
failure to thrive; abdominal distention; anorexia;
diarrhea; steatorrhea, that is the passage of
malabsorptive stools laden with fat; anemia; growth
failure; and vitamin deficiencies. That was really
the picture that we had of celiac disease 30 years
ago.
(Slide.)
DR. MURRAY: However, we now see celiac
disease in adulthood. In fact, celiac disease can
present at any age. Symptoms can include things
such as abdominal pain, even upper-GI symptoms:
heartburn, nausea, vomiting, anemia, fatigue.
There are of course patients who have
symptoms of malabsorption, though not necessarily
the classic, fully evolved malabsorptive picture.
Steatorrhea as a presenting symptom is relatively
rare, even patients may have constipation.
(Slide.)
27
DR. MURRAY: It can mimic other disorders
such as lactose intolerance. Indeed, lactose
intolerance may be secondary to the damage caused
by celiac disease. It may mimic the symptoms of
irritable bowel syndrome or symptoms of
inflammatory bowel disease.
(Slide.)
DR. MURRAY: There are specific
deficiencies that can occur in celiac disease,
especially the fat-soluble vitamins -- D, E, A,
and K -- with their resultant syndromes from
deficiencies.
Iron deficiency is especially common in
celiac disease because iron is absorbed in the
proximal small intestine; folate deficiency, again,
because it is absorbed in the proximal small
intestine; and, interestingly, B12 deficiency may
be relatively common in celiac disease by a variety
of mechanisms. Other trace elements -- zinc, B6,
selenium, and others -- may also be deficient in
celiac disease.
While in the past we would look for
28
combinations of these, often a patient would
present with many of these deficiencies at the time
of diagnosis, now it is relatively uncommon to see
the entire spectrum of deficiencies. Indeed, you
usually see one or two deficiencies that are
clinically evident, and the others may not even be
present.
(Slide.)
DR. MURRAY: How about non-intestinal? I
have mentioned that the major site of injury is in
the gut, but there are patients who will present
with non-intestinal presentations which can involve
into things such as the musculoskeletal system,
joint pains and osteoporosis or osteomalacia;
infertility or reproductive issues, delayed
puberty, spontaneous recurrent abortions have been
described; hematologic, which is predominantly
anemia; hyposplenism is an unusual consequence but
can present; and then dentition, enamel defects,
can be a presenting feature.
(Slide.)
DR. MURRAY: To focus on iron
deficiency
29
anemia, which is probably one of the most common
reasons that I see celiac disease, about 5 to 8
percent of adults who present with unexplained iron
deficiency in some studies have celiac disease.
It is especially common in those who are
resistant to the use of oral iron. If you look at
individuals who are coming to gastroenterologists
for endoscopy, it may be 5 to 15 percent of those
patients, depending on the study, who may have
celiac disease if biopsies are taken from the small
intestine.
However, many of those patients are even
missed because the biopsies are still not taken
during routine endoscopy in patients who have got
anemia in about a third to a half of patients.
(Slide.)
DR. MURRAY: Osteomalacia or bone disease,
this is an example of severe disease with
pseudofractures in the pelvis of an individual,
whose only presentation was osteomalacia with no GI
symptoms, caused by celiac disease.
Other non-intestinal presentations
include
30
neurologic or even neuropsychiatric syndromes such
as neuropathy, ataxia, seizures, cognitive decline,
or dementias; fibromyalgia-like syndromes or
chronic fatigue syndrome-like presentations;
individuals with skin and mucous membranes, there
is a specific rash associated with celiac disease,
a recurrent aphthous ulceration of the mouth; the
dental enamel defects we mentioned.
(Slide.)
DR. MURRAY: And, then, dermatitis
herpetiformis was specifically mentioned, because
this very blistering, extremely itchy skin rash
that affects the extensor surfaces is a direct
manifestation of intestinal gluten sensitivity.
(Slide.)
DR. MURRAY: Now, what about other
associated conditions? Celiac disease is
associated with other autoimmune conditions. It
may be seen in 3 to 7 percent of Type I diabetics,
individuals with thyroid disease, individuals with
inflammatory arthritis, primary biliary cirrhosis,
as examples
of others; and then the congenital
31
disorders, especially those associated with
chromosomal abnormalities and also selective IgA
deficiency. If you look at relatives of celiacs,
it is anywhere between 5 to 20 percent, depending
on how one is related to someone with celiac
disease.
(Slide.)
DR. MURRAY: Now, beyond the symptomatic
celiac disease, there are also individuals who have
no symptoms, who already have fully evolved damage
within their intestine, and there may be no symptom
or there may not be occurring in someone with an
associated disease.
This is frequent to find this in first-
and second-degree relatives of patients with celiac
disease.
(Slide.)
DR. MURRAY: There is also what is termed
"latent" -- well, whether it is latent celiac
disease or latent gluten sensitivity -- individuals
who have a positive serologic response but have a
negative
small-bowel biopsy. Some of those
32
patients will go on to develop the full-blown
disease, if followed, on a normal diet.
(Slide.)
DR. MURRAY: What about the epidemiology?
To summarize, while it was first identified in
Europe, it occurs essentially in all populations,
which could be termed Caucasian. Its prevalence is
probably somewhere between 1 in 90 to 1 in 300;
however, the diagnosis rate is much lower, which
would suggest a prevalence of about 1 in 2,000, if
you just look at the diagnosed cases.
It is one of the most frequent genetically
based diseases. If you look at other countries --
Latin America, or other areas; Africa, especially
North Africa; if you look at Asian countries --
there is celiac disease present in those. The
worldwide average prevalence is somewhere very
close to 1 percent.
(Slide.)
DR. MURRAY: I'm coming a little closer to
home. This is the data from Olmsted County, that
we
published a couple of years ago, which looked at
33
the new case identification or the incidence rate.
The solid yellow line is the new cases per
100,000 in the population, which is essentially
quite low and stable over many decades until the
1990s and into 2000 to 2001, showing a greatly
increased rate of detection of celiac disease.
If we looked at who were being diagnosed,
this is the age of diagnosis by age category. This
is the incidence per 100,000 of people in that age
category in the community. You can see that the
new cases being diagnosed are predominantly people
between the age of 45 and 64.
The solid line indicates females; so,
females are diagnosed at a rate about twice that of
males of all ages.
There are a significant portion of
individuals, almost a third, who were diagnosed for
the first time over the age of 65.
(Slide.)
DR. MURRAY: One can term or consider
celiac disease like an iceberg. These are a series
of icebergs
where the tip of the iceberg is the
34
part that has been detected, and the part
underwater is the part that can be found if one
screens the population. Of note, these are numbers
per thousand not percent.
Obviously, there are some places like
Ireland, for instance, which is quite a big iceberg
with a lot above water but also a lot below water.
Finland, which this iceberg is probably even more
out of the water as they have got a very active
program for finding celiac disease.
This is circa 1996. The U.S.A. iceberg is
still close to a very low level of actually being
diagnosed; but this number, the part underwater,
has actually grown to be something very close to
what you would find in Finland or Ireland,
especially when one looks at, at least what we know
about is really from a Caucasian population.
(Slide.)
DR. MURRAY: One miniature study, this is
one we simply looked at, Natrona County in Wyoming.
This is a very isolated community. Anybody who has
been to
Wyoming, there is lots of nothing for miles
35
and miles.
We were able to study about 4,000
individuals from a health fair, generally healthy,
and found the numbers above just under 1 percent of
people who had serologic evidence for celiac
disease.
Only half of them had GI symptoms. Most
of them did not have other risk factors for celiac
disease, just two having family members with celiac
disease.
However, these are numbers that would
confirm basically the rest of the world's data that
suggests that the prevalence of celiac disease, if
you look for it, is probably slightly under
1 percent.
(Slide.)
DR. MURRAY: Now, how does one make the
diagnosis? It starts with suspicion. Serologic
tests may be very effective at detecting the
disease. The intestinal biopsies are regarded as
the gold standard. Then, one ultimately gets a
response to
a gluten-free diet to confirm the
36
diagnosis.
(Slide.)
DR. MURRAY: The pathology, as we have
mentioned already, is a pathology of chronic
inflammation within the intestine with features
such as intraepithelial lymphocytes on the surface,
villus atrophy or the loss of the villus surface,
great crypt hyperplasia, and then characterized by
being a lamina propria filled with lymphocytes,
macrophages, plasma cells, and even eosinophils.
(Slide.)
DR. MURRAY: There is however a spectrum
of damage that occurs, typified here by the Marsh
classification. This is classic disease, but there
are also milder forms of the disease that may be
asymptomatic in most individuals.
(Slide.)
DR. MURRAY: Our algorithm for finding
celiac disease, if we have a high clinical
suspicion, an individual with malabsorption, we
biopsy those individuals. If we have an individual
who is at
moderately increased risk, serology is
37
probably the most effective way of finding it.
Though, we yet do not depend on the serology alone
to detect this, there are other circumstances for
alternate; serologic testing may be necessary.
(Slide.)
DR. MURRAY: Now, what about treatment for
celiac disease, or, as one patient with celiac
disease called it, "playing gluten-free roulette"?
(Slide.)
DR. MURRAY: The nuts and bolts of a
gluten-free diet, basically one needs to avoid
foods that contain the offending grains: wheat,
barley, rye, and the wheatlike grains of spelt and
kamut.
I put down at the bottom that many corn or
rice commercial cereals do not appear to be
gluten-free because of their incorporation of
particularly barley extract in their flavor
ingredients.
(Slide.)
DR. MURRAY: Now, one of the issues and of
course the
issue for today is, How much gluten is
38
too much? We will be hearing a lot more when you
hear prospective and retrospective data later this
morning on this. I am not going to dwell on it.
One thing from clinical appreciation is
that symptoms are not a good indicator of gluten
ingestion. Many patients can have significant
damage to their intestine, despite the absence of
symptoms when they ingest gluten.
Most patients diagnosed clinically with
celiac disease have never suspected that wheat or
gluten products are what are precipitating their
symptoms. They may have or are often likely to
have blamed other foods that they weren't able to
digest because of the damage.
Antibodies such as tissue transglutaminase
antibodies are really only positive of the
substantial gluten contaminating the diet. At
least in my practice if somebody admits to cheating
more than once a month they will like continue to
have injury in their gut. However, there is a high
degree of variability in the sensitivity to gluten
ingestion,
at least clinically.
39
(Slide.)
DR. MURRAY: We will hear a little bit
about Codex Alimentarius draft standards. This,
however, is still a draft, I think a Stage IV
draft. I want to put this up really to demonstrate
that there is a variance between countries and what
one allows.
We will hear more from our colleagues from
Europe about some of the incorporation of rendered
gluten-free foods which use the gluten-containing
grains as a base and then remove the proteins from
them.
(Slide.)
DR. MURRAY: What about non-responsive
celiac disease? This is relatively common. By
far, the most common reason is inadvertent gluten
contamination of the diet and lymphocytic colitis,
pancreatic insufficiency, bacterial overgrowth, and
then only a few patients have true refractory
disease that no longer responds to exclusion of
gluten from the diet.
(Slide.)
40
DR. MURRAY: There are many potential
sources of contamination of the diet with gluten,
which include of course commercial cereals, eating
out, communion wafers, lipstick, airborne flour or
starch in certain work situations, so-called "soy"
sauces made from wheat, mislabeled or unlisted
ingredients have been an issue, and at least
allegedly some medications.
(Slide.)
DR. MURRAY: Some ingredients that people
are concerned about: seasonings and spice blends,
modified food starch, malt and malt extract,
modified hop extract or yeast-malts, sprout
extract, dextrins, caramel color. There are a
whole bunch of things that might be derived from
gluten-containing grains.
(Slide.)
DR. MURRAY: What about complications
associated with untreated celiac disease? We know
the mortality of symptomatic celiacs who do not
comply with the diet has doubled. The mortality of
celiac
disease even when it is diagnosed is also
41
double, even following out for several years after
the diagnosis.
The predominant excess in death comes from
GI malignancies. There are also morbidity
consequences such osteoporosis or osteomalacia,
stunted growth, infertility, chronic ill-health --
all of which could be prevented by early detection
and treatment.
(Slide.)
DR. MURRAY: What are the dangers of
non-compliance? The increased mortality we
discussed, the osteoporosis. Children who were
diagnosed and then did not remain on a gluten-free
often have osteoporosis or diminished bone density
when they get to adulthood, lymphoma, other
cancers, and then the psychological effects of
non-compliance.
(Slide.)
DR. MURRAY: One of the most feared
complications of this are the T-cell lymphoma.
This is celiac disease on this side, and the T-cell
lymphoma on
the other side is one of the more
42
feared complications with a very high mortality.
(Slide.)
DR. MURRAY: However, as I pointed out,
most celiac disease is probably not symptomatic, at
least when we look at a cross-section of the
population. We do not know whether those
identified by screening are less sick than
clinically diagnosed celiac disease.
We don't know the benefit or negative
effects of a gluten-free diet in those who are
found by screening alone. We don't know if they
are any more or less likely to comply with a
gluten-free diet. We don't know whether
intervening in those patients will actually affect
their ultimate mortality.
(Slide.)
DR. MURRAY: George Dennison Prentice
said, "What come call health, if purchased by
perpetual anxiety about diet, isn't much better
than tedious disease."
(Slide.)
DR. MURRAY: This comes to the
future.
43
There is, I think, a promising future in celiac
disease, a variety of approaches, which I have
listed, that individuals and research groups are
looking at as alternates to the gluten-free diet,
though none of them are really even close to
clinical use.
(Slide.)
DR. MURRAY: This is one that has been
tested in patients using a lactobacillus digestion
strategy, trying to reduce the potential, harmful
effects of gluten.
In summary, I would like to suggest that
gluten or celiac disease is common. It has been
largely unrecognized until recently. There are
many challenges that face patients and their
physicians in the treatment.
The gluten-free diet is not simple. There
is widespread use of grain proteins in good, and
that makes it challenging for individuals with
celiac disease. Food ingredient source
identification is of great concern to patients.
Dietitians and those who counsel patients
44
with celiac disease, we are here because of
regulation or potential regulations. Defining
acceptable thresholds and verification of those may
be very important to patients with celiac disease.
(Slide.)
DR. MURRAY: I finish with an aside on
another food safety issue, an invitation to come to
Minnesota and enjoy Joe's and have worms at the
same time.
Thank you.
CHAIRMAN DURST: Thank you very much,
Dr. Murray.
QUESTION-AND-ANSWER SESSION
CHAIRMAN DURST: I would like to ask the
Committee if they have any questions or discussion,
and also to point out that Dr. Murray will not be
able to stay around for discussion this afternoon.
If there are questions, now is the time to ask
them.
Doug.
DR. HEIMBURGER: Thank you very much for
that
presentation. It is very helpful. I do care
45
for some patients with celiac disease. As you
already mentioned, one of the big questions that
they have is, "What really are my risks if I play
with it a little bit? If I knowingly introduce a
little bit of gluten in my diet, how absolutely
obsessive do I need to be about it?"
With the mortality being primarily
associated with the risk of lymphoma, what is the
quality of the evidence that compliance with gluten
restriction is really correlated with the risk of
lymphoma?
DR. MURRAY: The evidence, the quality of
the evidence is largely observational, taking
cohorts and following them, and often their
self-assessment of their level of compliance. The
data is based on those cohorts. They are
predominantly referral cohorts.
The longest follow up comes from Britain.
In those, there is quite a clear increased risk not
just for lymphoma but other malignancies in those
who are considered not to be completely compliant
with the
diet. I would say the level of evidence
46
while it is not a prospective study, in retrospect
in fact it appears to be reasonably strong.
DR. BRILEY: Margaret Briley. I would
like to know what you can tell us what might be the
reason for the increased detection of celiac
disease? Is the serological test the definitive
reason? What is going on with those? It seems to
be more prevalent, as you said, in our society.
DR. MURRAY: I think there are probably
two reasons, one is probably serologic detection
has made this an acceptable diagnosis to primary
care physicians, number one; and the second is
suspicion, that is, the awareness of celiac disease
as a possibility.
I do not believe, however, we can rule out
the possibility that celiac disease is actually
increasing in prevalence over time.
We don't know, but we know that there
certainly were substantial increases in other
inflammatory bowel diseases over the last 50 years,
which may not have been accounted for by detection
rates, for
example.
47
I do agree with you it is probably the
sero-detection. It is a combination of those two
things, suspicion and ease of detection have made
the biggest difference. However, I would not rule
out the possibility of its actually increasing in
prevalence.
CHAIRMAN DURST: Soheila.
DR. MALEKI: Soheila Maleki. I was
wondering how long does a person that is
asymptomatic go before they find they are
symptomatic? How do they usually find out?
DR. MURRAY: First of all, we don't know
when it starts. There is one study I didn't talk
about from Denver that suggested that children, if
you follow children at genetic risk for celiac
disease based on HLA type, that they will convert,
sero convert, by about the age of seven, though
most of them are asymptomatic.
It reaches all but 1 percent of that
childhood cohort, the same prevalence you find if
you look at adults. The suggestion is that you
have
developed that celiac autoimmunity probably by
48
the age of seven.
However, the age, the median age, of
diagnosis is 45 years of age. It is likely that
those individuals have some clinical disease for a
long time before they present.
In our population, the most common reason
for presentation is postmenopausal anemia. Women
who have anemia sometimes go a long time,
especially when they are menstruating. The doctor
says that menstruating is an excuse for the anemia.
Now that they are menopausal and are no longer
menstruating they no longer have an excuse for
their anemia, and they are referred for evaluation
for the cause of anemia.
Iron deficiency anemia at least is one of
the most common, but there are other things. For
example, the development of chronic GI symptoms.
Largely, as the earlier questioner said, probably
awareness of the possibility of this disease, with
primary care doctors using sero-diagnosis to find
celiac disease.
There a lot of atypical symptoms.
There
49
are reservoirs of celiac disease, the Type I
diabetics, the family members of celiacs, those
with chromosomal disorders. As the doctors looking
after those people become more aware of celiac
disease, they are testing their patients more.
DR. BRILEY: Could you speak a little bit more about
the evidence that you have in regard to physicians
using the serological test? Is it a pretty common
thing to do it at the very beginning of a patient's
coming in with GI problems, or is it more likely it
is down the road a while?
DR. MURRAY: If you look at the data on
patients’ presentation time, from presentation to
diagnosis of celiac disease, it is somewhere
between 8 and 11 years from the time they present
to their physician with a complaint to the time
their diagnosis is made.
I think that time period is now beginning
to shorten, thankfully. The serology is done close
to the end of that period, so it is often only when
the suspicion is generated. Partly that is because
it is not
considered at the early differential
50
diagnosis of celiac disease.
It is also difficult. It is difficult
because the symptoms are not very specific.
Fatigue and some bowel disturbance and a little
anemia, that is not a rare syndrome or not a rare
collection of symptoms you will see in individuals
in our community. It is hard to know where you
look for celiac disease and when you look for it.
It is often a very delayed diagnosis.
DR. BRILEY: Is the serological test one
that is pretty accurate? Could one count on it if
you did it early?
DR. MURRAY: If you use the more modern
autoimmune tissue transglutaminase test, for
example, it is reasonably good. It is not perfect,
but it is quite efficient at detecting celiac
disease at that level, in that period. It is not
perfect, though, but it is a pretty good test.
DR. BRILEY: It sounds like we need to do
some education, then.
DR. HEIMBURGER: A follow up to that --
Doug
Heimburger -- what is currently understood to
51
be the sensitivity specificity of anti-tissue
transglutaminase antibodies?
DR. MURRAY: Depending on the study, most
studies suggest that the sensitivity of tissue
transglutaminase for celiac disease is in the high
90 percent, so the high 90s.
The specificity is probably also in the
mid- to high 90s, so quite effective when you are
looking at a population you are suspicious of the
disease.
DR. BRILEY: One more question.
Margaret Briley. Do we have any data that shows
the celiac patient also maybe has been identified
with a lactose intolerance? Is there anything that
combines those two that you would know or does
that--?
DR. MURRAY: The combination is probably
the damage that is caused by celiac disease, the
damage to the entrocytes affects your disaccaridase
including lactase throughout the surface of the
small bowel, and, hence, you get a secondary
lactose
intolerance.
52
In general, if you think of the genetic
basis or the ethnic groups that are involved,
celiac disease tends to hit those of European
extraction more so. I know that population will
tend to retain their lactase activity for longer.
If you look at Subsaharan Africans, for
example, we don't know actually what the prevalence
of celiac disease is in Subsaharan Africans, but
they are individuals who tend to lose their
lactase.
Genetically, they are probably separate, but they
come together because of secondary lactase
deficiency caused by the damage in celiac disease.
DR. NELSON: Thank you, Dr. Murray. That
really was a very interesting presentation. I
think the last time I studied this was 30 years
ago.
CHAIRMAN DURST: Identify yourself,
please.
DR. NELSON: Mark Nelson. I wanted to
touch base on the couple of slides where you talked
about how
much gluten is too much and the proposed
53
Codex definition for gluten-free foods.
You had a question mark after the
threshold for damage being 20 to 100 milligrams per
day, and I guess that is similar to the naturally
gluten-free foods the Codex proposed.
Then, it goes on to talk about cheating is
greater than once per month. Also, is there an
issue of cumulative exposure, 20 milligrams per
day, or is the cheating an excursion of whole wheat
crackers, for example?
DR. MURRAY: Well, first of all, the
comment on cheating that is what the patients tell
me, those patients who will admit to gross
cheating, that is, eating a piece of bread or a
cookie or cake, which is what I regard as an
obvious source of gluten.
However, that may reflect some other
background, but a lack of detail of care, for
example, attention to detail in the rest of their
diet, maybe what they are not telling us. That
only applies to what they have told me.
The issue of actual threshold, I have a
54
question mark after that because we will hear a lot
more detail, science, about threshold testing for
thresholds of gluten contamination.
The intermittent contamination, once a
month obvious contamination. Something that is, to
some degree, under the patient's control with
appropriate education and exposure to information
then essentially it is under their control.
It is the low-level contamination on a
regular basis from sources they are not aware of,
and those are the patients that I see that make up
the majority of the patients I see who have
difficulty. They are coming to me because they are
trying to be gluten-free, but they are having
contamination of their diet on a daily basis,
probably a relatively small amount. We will hear a
lot more about the threshold, the actual testing of
the threshold using that type of low-level daily
contamination.
DR. MALEKI: Soheila Maleki. Is there an
adult onset or spontaneous development of celiac
disease, or
does this have to come with a genetic
55
component and hereditary?
DR. MURRAY: It probably only occurs in
people who have that HLA or genetic type. You have
to have it. However, that is 30-plus percent of
the Caucasian population.
Can it start first in adulthood? We have
very little data on that. The only data is the
stuff I have mentioned, looking at children and
showing about 1 percent by the age of seven or
eight, positive by the age of seven or eight, the
same prevalence if you look at adulthood.
There are a few cases of what we call
"latent celiac disease," individuals who have got
antibodies with apparently normal small intestinal
biopsies as adults, then go on over a space of
years to develop full-blown celiac disease within
years of that initial identification of a positive
serology. Those are very rare cases that have been
found.
Of course, if you find somebody who
suspects they have got a problem, they ought to
change
their diet anyway, and that changes
56
everything. There is very little data, I think, to
be sure of whether it first occurs, starts, in
adulthood.
If you look at the age of diagnosis, your
eighth or ninth decade can be the first time that
you are diagnosed with celiac disease. A lot of
those patients have suspicious symptoms going back
many years.
DR. BRITTAIN: Erica Brittain. Can you
quantify the level of damage? Is that only
possible with the biopsy, and is that very
invasive?
DR. MURRAY: You can -- well, if you take
biopsies, you can quantify the degree of injury. I
showed that slide which shows a spectrum of injury.
It tends to be variable, even within the same
individual. It started in the first part of the
small intestine and extends a variable distance
down the small intestine.
There has really only been one study,
which was done in the early 1960s, of taking
multiple
biopsies down the intestine -- a handful
57
of courageous volunteers.
It is not clear that there is a
correlation between the extent of injury and the
severity of symptoms. In fact, we don't know how
to predict the occurrence of symptoms in patients
with celiac disease, so that is a "black box."
DR. BRITTAIN: You are saying there is
really no simple way to quantify the extent of
damage? There is nothing that would correlate with
these multiple biopsies?
DR. MURRAY: Some people have suggested
that the level of the antibodies, the tighter the
antibodies might correlate: The higher, the
tighter, maybe the more severe the injury to the
intestinal biopsies.
When we take a biopsy of the intestine, we
are sampling a tiny fraction of a percentage of
1 percent, a fraction of 1 percent, of the
intestinal lining. We have tried to look using
other imaging techniques to assess the extent of
injury. Using some of those techniques seems to be
very
variable between individuals, and it doesn't
58
seem to predict their symptoms.
Really right now it is a yes or no issue:
yes, they have celiac disease; or, no, they do not.
It is very hard to measure the severity of the
disease.
We can look at the severity of
consequences: have they developed osteoporosis,
what their bone density is, have they lost a lot of
weight, whether they have severe malabsorption
based on fat malabsorption in their stool. We have
got other measures to look at the consequences or
impact of the disease, those we can assess.
DR. BRITTAIN: Do you think that it would
correlate with the intestinal damage? Not
necessarily?
DR. MURRAY: We have tried. I would say
that the data is not very good on that. People
with very mild injury may be more likely to be
asymptomatic, but the data is not sound.
CHAIRMAN DURST: Suzanne.
DR. MALEKI: Suzanne Teuber. I had a
question
about the neurologic presentations. I
59
have read some on screening of pediatric
populations, but with adults how often does it
present as dementia without it being diagnosed as a
GI problem? Is this something we should be adding
to dementia screening?
DR. MURRAY: It is probably relatively
rare. In fact, I think there has only been one
good study. Maybe Dr. Collin, who is here as a
speaker later, has done a study and has looked at
and reported on that.
We occasionally see cognitive decline at
the time of diagnosis, but there is really no good
epidemiologic study to address that. Some of the
other presentations, peripheral neuropathy, for
example, or ataxia, there is more data on it. Many
neurologists are beginning to include celiac
disease as part of their differential diagnosis for
those syndromes. Good epidemiology data is
relatively small, very little data.
CHAIRMAN DURST: Ciaran.
DR. KELLY: Ciaran Kelly. This is
actually
more clarification than a question. Dr.
60
Murray is quite correct that there isn't a measure
of either severity of intestine abnormality or even
height of antibody levels that reliably reflects
the degree of injury or correlates closely with
symptomatology.
However, with treatment one can use a
decline in antibody levels as a crude indicator of
at least reduced exposure to gross amounts of
gluten. It is not a very sensitive indicator, but
it is useful. Of course, with repeat biopsy, if
the histology has revered to normal, that of course
can be used. However, the less invasive test of
following antibody levels is used clinically to
follow response.
DR. MURRAY: Quite right. I think you
will agree, Joe.
DR. KELLY: If the antibody levels aren't
dropping, that is used as an indication that the
patient is successfully on a gluten-free diet.
DR. BRILEY: Margaret Briley. Can you
give us any idea of any behavior data that you may
have
received from your patients regarding their
61
willingness to try foods that are not gluten-free
labeled?
DR. MURRAY: Oh, well, there are many
different attitudes among patients with regard to
what they want to eat or what they are afraid to
eat.
I advise my patients to be prudent, that
they try to select things based on identification
of ingredients, source ingredients, not containing
things contained from gluten, the use of substitute
grains that are gluten-free.
Many patients are quite willing to do that
on their own. Many of them use support group
information where maybe a group has cooperatively
contacted manufacturers who in good faith provide
information on their source ingredients.
There are some patients who are entirely
paranoid about it, and want to obtain a kit to test
the food. I don't know that we've got a very
effective kit yet for testing food for gluten
contamination. There are many different attitudes.
Fear is a major concern among my patients.
62
I mean, fear of even the slightest potential, not
even actual but potential contamination. This can
verge on, "Do we avoid taking prescription
medications for things like hypertension?"
resulting in life-threatening changes to their
medication regimens because of fear of
contamination.
I would say fear is a major part or a
major influence on the quality of life. We will
hear more I think shortly on the impact of a
gluten-free diet on patients' lifestyles a little
later. Certainly that does affect a substantial
portion of my patients.
Patients go through a substantial grief
reaction and feel socially isolated because of
their difficulty of interacting with society,
because so much of our society activities or social
activities revolve around food. There is that
safety sense of insecurity, which I think pervades
or affects many patients with celiac disease.
DR. BRILEY: Thank you.
CHAIRMAN DURST: Soheila.
63
DR. MALEKI: Well, I just want to know if
there are any coordinated studies for a
determination of thresholds? I know you mentioned
the level of PPMs. Do you know of any studies?
DR. MURRAY: There are and you will hear
about them. There are both retrospective and
prospective studies, and you will be hearing some
data on those later this morning.
CHAIRMAN DURST: I have one question --
Dick Durst -- on the biopsy and histological
studies to see the morphology, morphological
changes, you showed from the shag rug. I am just
curious whether just one of these cameras you can
swallow would be able to detect those kind of
changes without having to go through a biopsy?
DR. MURRAY: Yes, you can detect them.
Nobody would suggest that it would replace the need
for biopsies to make the diagnosis. There is
really relatively little published data on it.
There has been a paper suggesting that you can see
those changes. With a magnified view, you can see
with a
capsule.
64
Yes, I think you can identify those
changes in a lot of individuals with celiac
disease, maybe all of them. Although, I really
can't comment more on that, because it hasn't
really been studied in any great detail.
CHAIRMAN DURST: Erica.
DR. BRITTAIN: Erica Brittain. Do you
have any insight about the cumulative effect of
decades of low levels, very, very low levels of
gluten exposure? Certainly, we are going to have
to think about what chronic exposure could do when
we talk about the thresholds. Can you provide any
insight into that?
DR. MURRAY: Probably the best clinical
insight I can give are individuals who I see who
were diagnosed 20 years ago and have not come back
to medical attention in 20 years. I see those
patients maybe every week.
I would see somebody who is diagnosed 20
years ago, and they got instruction at that time
that allowed them to eat things like barley malt or
that people
weren't really instructed about some of
65
those rice or corn cereals that may have contained
malt, for example.
Those patients come back with anemia,
chronic GI complaints, maybe not as severe as they
had initially, but they certainly have accumulated
some health morbidity over those 20 years. Some of
them will come back with frank lymphomas and will
end up with a mortal complication of their celiac
disease.
Yes, at least my clinical observation is
that I frequently see individuals with problems
that we get rid of, once they now move to a much
more strict gluten-free diet, by eliminating those
things -- largely, because in 20 years they didn't
go back and get more education and realize that you
had to exclude those minor ingredients. That is
one way of looking at the effects of decades'
accumulation of low-level contamination.
CHAIRMAN DURST: Dick Durst again. On
your slide that showed the various causes, the
different grains, and so on, I believe you
indicated
oats was not one of the causes. Could
66
you expand on that?
DR. MURRAY: We will hear a little more, I
think, from Dr. Collin on that issue. While oats
had been thought to be one of the offending grains
in things done in the fifties and sixties, it turns
out from recent very well-done studies that it
doesn't appear to impair the healing of the
intestine in newly diagnosed celiac disease. It
doesn't seem to result in a significant worsening
of production of damage in patients who are already
diagnosed with celiac disease.
For the vast majority of celiacs, it is
probably safe in its native, pure form. However,
there are some sequences within oats that can
produce an immune response, at least in vitro, in
lymphocytes derived from a few celiacs.
It is not an absolute. There may be some
individuals with celiac disease that can respond to
oats, both in the laboratory test and possibly also
clinically there are a few.
There are probably a relatively small
minority of
celiacs in which that occurs. A bigger
67
concern is the issue of contamination of oats with
other grains that are well recognized to cause
injury.
CHAIRMAN DURST: Marc.
DR. SILVERSTEIN: Marc Silverstein. I
would like to inquire about the potential
subsequent lifelong increased risk of GI cancers.
I presume that the risk is predominantly small
bowel, but I wonder if there is any increased risk
of colorectal cancer?
Then, what are your thoughts about whether
there is sufficient risk that patients with celiac
disease should be in some sort of surveillance
program for early detection of GI cancer?
DR. MURRAY: Clarifying the risk of
cancers, it is particularly visceral cancer but
also includes: esophageal cancer, non-Hodgkin's
lymphoma of any site not just the intestine, and
probably also B-cell lymphomas, as well as the
T-cell lymphomas, small-bowel carcinoma.
There is a greatly increased relative risk
of
small-bowel carcinoma. Of course
small-bowel
68
carcinomas is a very rare disease to begin with, so
the lifetime risk of dying of a small-bowel cancer,
even in a celiacs, is still relatively small. The
data on colon cancer is mixed. There is some that
suggests there is an association; and some, that
does not.
When you look at other causes of
mortality, even non-cancer causes of mortality such
as infections, neurologic disorders and chronic
lung infections, there are other excesses of
mortality that occur in patients with celiac
disease.
There are some reductions in cancer
mortality. It appears, at least there is a
suggestion, that breast cancer may be less common
in celiac disease than non-celiac disease. There
were a couple of suggestions that lung cancer might
be less common in celiac disease than in non-celiac
disease.
Now, whether there is some competing issue
like smoking may be less common in celiac disease
than
non-celiac disease, so there may be some other
69
competing issues that are involved. Body size may
make a difference. It may be another confounding
issue that confounds or is a competing risk for
malignancies.
While small-bowel cancers and lymphomas
are the two that have the greatest relative risk,
it is a small, absolute risk because of the
relative rarity of those cancers.
CHAIRMAN DURST: Do we have any further
questions for Dr. Murray?
(No verbal response.)
CHAIRMAN DURST: Thank you very much.
Our next speaker is Cynthia Kupper, who is
the executive director of the Gluten Intolerance
Group of North America who will present on patient
perspectives on celiac disease.
PATIENT PERSPECTIVES ON CELIAC DISEASE
MS. KUPPER: Good morning. I am a
dietician, not a doctor. I appreciate the honorary
doctor status.
My job here today is to give a face for a
person with
celiac disease. I have been tasked
70
with letting you know who they are, letting you
know how they get their information and education,
and then also providing you with some information
about labeling that they have.
(Slide.)
MS. KUPPER: First of all, living with
celiac disease is very difficult. It is a chronic,
lifelong condition, as you have heard, and people
find this to impact greatly their quality of life.
Forty-four percent of the patients in a
Canadian study say that the diet is very difficult
to follow. In fact, there are some studies that
suggest that the compliance with the diet can be as
low as 50 percent in teenagers but probably ranges
around 70 percent compliance.
Eighty-four percent of these patients in
Canada suggested they have a difficult time
determining what is gluten-free and what is not.
They don't travel, and they don't eat out. It
impacts their family life and their career.
If you have celiac disease and are an
X-ray
technician, oftentimes you change careers
71
because sometimes the X-ray slides are dusted with
flour. Chronic exposure could impact your quality
of life.
(Slide.)
MS. KUPPER: I did a study online of 620
patients a few months ago. In response to that
study, 75 percent of them said that they can tell
the difference between a gluten reaction and other
intolerance or a food allergy.
When they discussed their reaction
symptoms, they ranged from anaphylaxis, which is
not a gluten reaction for celiac disease, to
delayed reactions which could impact any aspect of
their GI and other health systems, body systems.
The average time to reaction was somewhere
between four to eight hours, but some of them
complained of immediate, almost allergic type
responses, and many of them said that their
responses or the symptoms that they had would last
for several days.
Keep in mind, there is no medication we
can give
them to make this go away, so they just
72
have to let it work its course. This is really
disturbing to me.
(Slide.)
MS. KUPPER: As a dietitian, patients do
not rely on medical communities and professionals
for their information. They rely primarily on
support groups.
Actually, the Internet should probably be
the first one, because they are Internet savvy.
They have been out there and they have gotten all
kinds of information before they ever see a
dietitian. Not only do they get information from
the Internet and the support groups, but there are
list serves and chat groups that they belong to.
These can be very useful tools for a
person with celiac disease. However, they also
provide some very frightening and unreliable
information that the patients will hold onto as if
it were gospel. Then, they work with self-help
books as well.
Unfortunately, doctors, and especially
dietitians,
are seen as unreliable. It is sad for
73
me to say that as a dietitian my profession doesn't
get this disease. They also treat it like it is a
rare thing, and they don't know anything about it.
In the United States, I can tell you that there is
probably a handful of dietitians who would be
considered experts in celiac disease.
Doctors don't get much more respect,
primarily because it has taken so long for the
patient to get a diagnosis that the patient has
lost faith.
Then, they will go to research facilities
like the University of Maryland, Chicago, and
New York, or the Mayo Clinic. Lastly, they will go
to medical Web sites. The bulk of our information
is coming from potentially unreliable and
non-research-based sites.
(Slide.)
MS. KUPPER: The consumers perceive that
gluten exposure levels -- the question was asked to
me, "What do consumers believe about gluten
exposure? Are they concerned about the health
risks?" The answer is yes
and no.
74
On the study that I did, it depended upon
their confidence of the labeling, and it depended
on whether they accepted testimony or accepted
research. There is a group of celiacs, as
Dr. Murray suggested, who really don't want to
listen to what research suggests.
As you move forward, with not only
establishing how you are going to determine the
threshold but what that threshold will be, you will
have a fight in the celiac community for a lack of
education and understanding of research.
Consumers oftentimes also have an
inability to correctly interpret research findings.
These are people who have just enough medical
knowledge to be dangerous, so they don't have a
full understanding of the terminology they are
talking about.
There is this constant perpetuation of
misinformation. I don't know how many times when
we try to bury something that is inaccurate it gets
dug up.
(Slide.)
75
MS. KUPPER: There are varying levels of
gluten sensitivity, as you heard, too. There is
the perception that gluten is poison. Not unlike
the allergy people that we heard from yesterday,
this is a huge issue to the celiac consumer.
They believe most of the time that when
their gut hurts it is from gluten not from
something else. Consequently, we are trying to
help the patient understand that not everything
that makes their gut hurt is gluten.
As Dr. Murray said, there is a huge fear
reaction. If I had to put a psychological label to
a group or at least a portion of the celiac
community, they are filled with fear and a little
bit paranoid about what they can and can't do.
How do we define "gluten-free" in the
U.S.? This is a really interesting question. Of
the consumers, only 19 percent realize that there
is no definition right now for gluten-free in the
U.S. Many of them define that the true definition
is zero. This is a problem -- a lot don't know.
(Slide.)
76
MS. KUPPER: When I ask the question, "Do
you trust gluten-free labeling?" It was
interesting, too, because most of the people say
they do trust it. However, when you ask them if
they ever had a reaction to a product labeled
gluten-free, you can see that up to 50 percent
suggested that they might have had a reaction to a
gluten-free product.
When I talk to manufacturers that
manufacture only gluten-free products and ask them,
"Do you test, and what do you test to," many of
them are using older testing methods not the newer
testing methods, the monoclonal tests that we
talked about yesterday.
Some of them tested 200 parts per million,
some of them tested 20 parts per million, some of
them tested no detectible. For the gluten-free
consumer today, the label "gluten-free" really has
no meaning.
(Slide.)
MS. KUPPER: Again, the gluten-free
consumer is
compulsive about their medical needs.
77
This is their only treatment. It is often referred
to as our drug of choice. There is nothing else we
can do, except to follow a strict gluten-free diet.
They have very limited trust in the
manufacturing industry. They believe that labels
that say "may contain" and different things like
that need to be distrusted. When they call the
manufacturers, they are not quite sure that they
are getting the right answer all the time.
Also, they have a limited understanding of
what good manufacturing practices really mean, so
they are always questioning what the manufacturer
will say. Yet, at the same time they want
accountability and they want reliability.
They may translate information to the
extreme. Let me give you an example. A few months
ago on one of the list serves, someone put out a
message about bottled water being gluten-free.
That got taken in a week's time to the
point where consumers were calling asking why water
had gluten in it, and how dare the food industry do
that to
them. The reality is it never did.
78
A company, out of the graciousness of
their heart, put it on a list of gluten-free
products, and from that the consumer decided that
every other bottled water had gluten in it. This
is the extreme that the consumer can go to. Again,
they don't find descriptive labeling helpful at
all.
The changes that can occur in ingredients
in manufacturing processes make it difficult for
this consumer group to know what they can have.
The term "modified food starch" usually means
cornstarch, modified cornstarch, in this country.
However, if the manufacturer determines
that wheat starch is cheaper in the fall and they
switch and the consumer has determined that this
product is gluten-free, now they are in trouble if
they don't recheck.
When you talk to the food industry, you
will find that their calls have dramatically
increased over the last 2 to 5 years of consumers
calling in, and 90 percent of the questions do not
have to do
with other allergens but have to do with
79
gluten.
(Slide.)
MS. KUPPER: When the consumer asks the
question about gluten, the problem is that they are
asking the wrong question. The consumers believe
that if they don't have effective labeling how can
anybody possibly know that they are going to be
able to be healthy and protect themselves.
They want to know that if you call a
company they are really giving you the right
answer, and they are just never confident about
that. Oftentimes, when the company answers too
quickly, they get suspicious.
Oh, I've had that experience. I will call
on a product and I'll say, "I need to know the
source of the modified food starch."
"Oh, you're talking about gluten?"
"Yes. Tell me the source of your modified
food starch. Let me make that decision about
whether I'm talking about gluten." That makes a
consumer suspicious.
Finally, you know, if a person eats a
80
gluten-free food and they get sick, whether it is
related to gluten ingestion or not, they have
determined that they can't trust that company any
longer.
Again, these list serves and chat groups,
I have seen them take small companies out of
business because of the spreading of rumors --
which are probably unfounded.
(Slide.)
MS. KUPPER: In closing thoughts, I really
encourage that through this entire process related
to labeling thresholds, that we be talking a common
language.
Let me use the example of threshold.
Yesterday, as I listened to Anne Munoz talk about
thresholds for allergens, I realized that we have
three different definitions of thresholds -- or
tolerance, excuse me. I want to use the word
tolerance.
The consumer says, "Tolerance is zero."
What that means is they think there should be zero
gluten in
their food.
81
The medical community says "zero
tolerance." For them that means, you should be on
a strict diet, and you should never cheat.
The manufacturing industry wants to know
where that is. Is it 20? Is it 200? They know it
is not zero.
We are not talking the same language. The
consumer needs to know that the manufacturer and
the industry or the legal ramifications around any
labeling are all using common terminology in a
language they can understand.
Education is a huge component. As much as
I am a supporter of this regulation and this law,
one of the things that is going to happen, as you
heard yesterday in discussions about soy lecithin
and other ingredients, it is going to become a bag
of worms. For the consumer, it is going to be very
confusing, and we need to have an education
component as part of the new labeling laws.
I encourage you, too, although you heard
it yesterday and you will probably hear it today,
too, we
know that there is no testing kit available
82
that tests to zero. We know, as you will probably
hear later, that it is probably an impracticality
or unnecessary to even go there.
I implore that when you set a threshold
method and testing methods, when you set the
threshold level, that it be reasonable and
something that meets the health needs of the
consumer but also allows the industry to meet the
need.
(Slide.)
MS. KUPPER: As they found out in
Australia, when you set zero as the tolerance level
and as the magic number for food manufacturers, a
lot of gluten-free products that patients used no
longer can be labeled gluten-free. Now the
consumer is once again confused and outraged.
CHAIRMAN DURST: Thank you very much.
Do we have any questions for our speaker?
Yes, Jeff?
DR. BARACH: Hi. Thank you. Jeff Barach
with Food Products Association. If I interpret
what you
said correctly, you were talking about the
83
consumer really doesn't find descriptive labeling
very helpful in the case of gluten-free.
I assume then the consumer would go to the
ingredient list or the 800 numbers or their
internal chat groups to find out whether the
product really is gluten-free or not. Am I
interpreting that right? Your constituency does
not want gluten-free labeling?
MS. KUPPER: I would say that is probably
right, that is the message I got from the survey.
In fact, they found that labels that say "may
contain" or "processed in a plant with" really is
frightening to them. They will look at a product
like that, and they will simply avoid it.
They do go to chat rooms and there are
lists of gluten-free products. However, when you
look at those lists and you ask how they were
developed, there are no standards for developing
those lists.
DR. HEIMBURGER: Doug Heimburger, a follow
up to that. That is not the same, is it, as saying
"gluten-free"? Do they
not want a label except it
84
is gluten-free with a consistent and clear
definition of that?
MS. KUPPER: They do want a label that
says gluten-free with a clear and consistent
definition.
DR. HEIMBURGER: Yes.
MS. KUPPER: I believe that gluten-free is
not is not going to mean zero; it can't mean zero.
CHAIRMAN DURST: Any further questions or
comments?
(No verbal response.)
CHAIRMAN DURST: Thank you very much.
CHAIRMAN DURST: Our next speaker is
Dr. Donald Kasarda, who is a consultant and retired
senior scientist from the Agricultural Research
Service of the USDA. He will make a presentation
on grains.
GRAINS
DR. KASARDA: Good morning everyone. I am
a research chemist retired from the U.S. Department
of Agriculture, although I still maintain a
relationship with my old lab in Albany, California,
85
as a collaborator.
Now, Dr. Murray covered a lot of the
things I am going to talk about. Maybe I will be
able to add a little bit more detail to some of
them, but he did an excellent job of talking about
some of the grain topics.
(Slide.)
DR. KASARDA: Immunology textbooks often
classify hypersensitivities into these four types.
Celiac disease is a delayed type hypersensitivity
that involves T-cells in the primary mechanism. It
falls into Type IV. Allergy is Type I and is
mediated by IgE antibodies.
Now, in the case of celiac disease, it is
often suggested that there is a Th1 mechanism
involved in which T-cells are presented with
gliadin peptides, and, ultimately produce
cytokines, inflammatory cytokines such as
interferon gammas as an example.
Now, in the case of allergy, however, the
same molecules that can induce the symptoms of
celiac
disease are also capable of producing
86
allergies. We do have a certain amount of
confusion sometimes between immediate
hypersensitivities and the delayed-type, celiac
disease.
(Slide.)
DR. KASARDA: Now, this is the same
diagram that Dr. Murray showed. I want to talk
about primarily the endosperm, which is this white
part here (indicating) in the cutaway diagram.
The starchy endosperm is made up of about
75 percent starch, but it also contains about 7 to
17 percent protein, depending on the use of the
wheat. Most of this protein, about 75 percent of
it, is gluten protein.
The proteins are storage proteins. They
are used by the developing plant that comes from
the germ here. The germ is separated from the
outer layers and the endosperm during the milling
process after crushing and sieving.
The storage proteins are broken down upon
germination of the seed to produce a new plant.
The
resulting amino acids and nitrogen are used in
87
the synthesis of new molecules needed by the
developing plant. Now, as I mentioned, about 75
percent of the storage protein is, in fact, gluten
protein.
(Slide.)
DR. KASARDA: This is a picture of flour
particles, a scanning electron micrograph. These
round, spherical structures are starch granules.
These (indicating) are A type, there are some
B types which are small here. The surrounding
rough-edged material is the gluten protein or
storage protein.
(Slide.)
DR. KASARDA: If you mix together flour
and water, as most of you have had the experience,
you can form a cohesive elastic dough. If you need
a dough under water, say, in a large container of
water or under a stream of water, you can wash out
the starch granules; they pop right out of the
matrix. You are left with a cohesive, elastic mask
consisting mainly of the storage or gluten
proteins.
88
(Slide.)
DR. KASARDA: Now, this is the traditional
cereal chemist definition of gluten. You cannot
carry out this process with rye and barley.
Therefore, to the traditional cereal chemist, there
is no gluten in rye and barley. However, the
celiac disease community has adopted the term
"gluten" for any protein that is toxic or harmful
to a celiac patient.
This terminology problem sometimes is
confusing when patients go to a company where they
might be dealing with a traditional cereal chemist,
and there is a certain amount of confusion as to
what is gluten. As I said, this is the traditional
definition, but it has been expanded to include
other grains that are harmful to celiac patients.
Now, from time to time, you will hear
about these fractions of gluten. Going way back,
at least over a hundred years, it has been
traditional to divide gluten into two, roughly,
equal fractions based on their solubility. This is
not an
exact separation. No solubility
fraction is
89
ever perfect.
Traditionally, it was alcohol-water
solution and sometimes we used detergent solutions.
We divide it up into the soluble fraction, which we
call "gliadin."
This is made up of monomeric proteins of
the prolamin class. The prolamin terminology comes
from Osbourne back around 1900. It is derived from
the fact that there are two major amino acids found
in the composition of these proteins. Proline and
glutamine, hence, prolamin.
By structure, we have three types: the
alpha type, gamma type, or omega types. Sometimes
people speak of the alpha/beta. I will talk about
that in a little as we go along.
Now, the insoluble fraction is called
"glutenin" by the cereal chemists. In rye and
barley, there is an equivalent fraction that we
called just generically "glutelin."
Now, this polymeric fraction consists of
prolamin subunits. Again, large amounts of proline
and
glutamine in the composition of the proteins.
90
These subunits are linked together by disulfide
bonds into a higher level of polymer.
Of course, a protein is a polymer in
itself. It is divided into two main types the
low-molecular weight and the high-molecular weight
glutenin subunits.
(Slide.)
DR. KASARDA: This just is a table showing
the percentages in the various types of proteins.
For example, you have the sum of glutamine and
proline ranging from about 40 percent up to about
80 percent in some of the omega gliadins.
This is pretty unusual to have such a high
percentage of glutamine and proline. This is key
to the toxicity, because the toxic sequences
involve glutamine and proline and usually an
aromatic as well, either tyrosine or phenylalanine.
(Slide.)
DR. KASARDA: Now, the terminologies that
we use really go back to early electrophoretic
studies in the late sixties and early seventies.
Again, if
we follow this diagram here, this is an
91
acid gel in which the proteins are separated by an
electric field in a polyacrylamide gel.
The terminology actually came from a sort
of free-boundary electrophoresis that was carried
out at our Northern Regional Research Center back
in the sixties. When they developed the
polyacrylamide gel electrophoresis, it was found
that the fractions fit with the mobility in the
electrophoretic gel.
You have the alpha, fastest moving; beta;
gamma; and omega. Structurally, the alphas and
betas are pretty similar. Some people will talk
about alpha/beta types. I just lump them together
as alpha types.
Now, the alpha type and gamma type are
about the same size. If you carry out SDS page or
polyacrylamide gel electrophoresis in detergent,
sodium dodecyl sulfate, which is a very good
dissociating agent for proteins.
Reduced or unreduced the gliadins give a
pattern somewhat similar to this. It is not quite
as good at
resolving alpha, beta and gammas as you
92
find in the acid gels where aluminum lactate was
one of the favorite buffers.
If we go over to the glutenin fraction,
and these are the subunits linked together by
disulfide bonds, if you try to take a purified
glutenin fraction and run it into an acid gel or
into a detergent gel, mostly you've just got a
little bit of streaking around the origin because
the polymers are too large to migrate into the gel.
(Slide.)
DR. KASARDA: Upon reduction, however, you
begin to see this type of pattern here in which
there is a group of high-molecular-weight subunits
and a group of low-molecular-weight subunits. This
only occurs for the glutenin fraction when you
reduce the disulfide bond.
(Slide.)
DR. KASARDA: This is a two-dimensional
pattern, electrophoretic pattern, of the gluten
proteins. All you have to recognize is that each
spot here represents a separated protein. There
are quite a
few different gluten proteins, and we
93
can count easily 50, 60, 70 spots in such a
pattern. Therefore, there are at least 50, 60, 70
gluten protein components.
We know from genomic studies that, in
fact, there are probably at least a hundred genes
coding for these proteins, and probably several
hundred genes coding for the proteins. The loci in
the genome that code for these proteins are spread
out over about nine different loci in the genome.
Now, as far as we know, all of these
gluten proteins are toxic in celiac disease. This
group here (indicating) are the omega gliadins, and
they seem to be particularly active.
However, all of the gluten proteins have
been tested by Paul Ciclitira's group and
Peter Howdle's group in the U.K., and they all
indicated by direct installation into the small
intestine that these proteins, all of these
different classes are toxic.
These omega gliadins are noted for being
strong allergen in exercise-induced anaphylaxis.
They are
one of the really strong antigens involved
94
in that particular allergy.
(Slide.)
DR. KASARDA: This is a schematic diagram
that illustrates the fact that all of these
proteins are noted for a repetitive domain in which
certain amino acid sequences are repeated over and
over again.
They are somewhat degenerate, but we can
derive consensus sequences. These are glutenin
subunits, gamma-type gliadins. These red,
staplelike lines indicate intramolecular disulfide
bonds.
In a glutenin subunit, we also have free
cysteines which can link up to another molecule to
form these higher-level polymers. For example,
here is an alpha-gliadin -- I'm going to talk about
this a little bit more -- the end terminal, or the
first half of the molecule, is made up of these
repeating sequences.
The second half is not repetitive and
contains most of the disulfide bonds. Toxicity
seems pretty likely to be limited to the
repeat
95
regions. These are the high-glutamine, the
high-proline regions.
Now, the omega-type gliadin seems to have
lost -- well, we are not entirely sure whether they
lost this type of domain or not, but in any case
they are made up almost entirely of repeating
sequences.
(Slide.)
DR. KASARDA: This is a hypothetical model
of the gluten polymer or glutenin in which the
subunits are joined by intermolecular disulfide
bonds, there are also these intramolecular
disulfide bonds, to form a higher-level polymer
that provides elasticity to a dough.
The gliadins and the glutenins are
cohesive with one another, but the gliadins
contribute more to the extensibility of the dough,
and the elasticity comes primarily from the
glutenin fraction.
(Slide.)
DR. KASARDA: Now, here I show some of the
types of
sequences that you find in the repeats.
96
Now, they look pretty similar, a lot of glutamine,
which is represented by "Q"; a lot of proline
represented by "P"; and usually an aromatic
residue, either phenylalanine, "F," or tyrosine,
"Y."
Somewhere, and these are often degenerate.
They are not exactly according to the consensus
that I show here. Somehow along the line these
sequences have acquired toxicity in celiac disease.
(Slide.)
DR. KASARDA: Now, this is the complete
sequence of an alpha gliadin. This is the
end-terminal region up here (pointing). It starts
at one, and there are 263 amino acid residues.
Here, note the predominance of the blue Q's and the
red P's for the proline and glutamine residues.
This half of the molecule here is the
repeat region. There is also this interesting set
of glutamines, which really hasn't been studied in
celiac disease. It is probably not toxic, but, as
I say, there has been almost no study of this
polyglutamine stretch here.
97
Note also these vertical lines here which
I show. Those are sites that we have observed
where cleavages occur with gastric enzyme, pepsin
and pancreatic enzymes, trypsin and chymotripsin.
Now, most proteins would be broken down by
the digestive enzymes into single amino acids or
very small peptides: diatride, tetrapeptides that
are easily absorbed, which are probably not toxic.
(Slide.)
DR. KASARDA: In the case, as Dr. Murray
mentioned, because we have a lot of proline which
interferes with the breakdown by the proteolytic
enzymes, we can get some pretty large stretches.
This stretch here from 31 to 55 right here is
something that we have tested as toxic.
Other people have dealt with sequences
from this stretch and found them also to be at
given toxic. The fact that this gliadin and
glutenin proteins are difficult to digest by the
digestive enzymes allows these toxic stretches to
exist longer than you would find for other
proteins.
98
Now, this half of the molecule has a fair
amount of glutamine and proline, but as far as we
know toxicity does not reside in this C-terminal
half or sort of the end of the molecule. It is in
sort of the forward end of the molecule.
(Slide.)
DR. KASARDA: Now, this is what I would
call my string of beads model in which I have just
taken that sequence, 1 to 263, and shown it as
beads on a string.
Each bead represents a different amino
acid. I tried to assign the different amino acids
different code words to distinguish them. This is
the end terminal region of repeats. This
C-terminal where we have the disulfide bonds here.
Toxicity resides in this part here.
This sequence here from 31 to 43 was
synthesized by Mike Marsh in the U.K. first, and he
instilled the synthetic peptide directly into the
small intestine of several celiac patients and
found changes in the mucosa that were indicative of
celiac
disease. So this does seem to be a
toxic
99
sequence.
Here I show a computer molecular model of
what that sequence would look like in the
polyproline II left-handed helical confirmation
that Dr. Murray mentioned. We think that these
peptides do have a strong tendency to assume this
polyproline II confirmation.
Here, I show the sequence in three-letter
code as a string of beads model and here just a
single-letter code. I know most people are not
used to dealing with these codes. I apologize for
using them in some of the slides, but often I am
just trying to make a general point, and you don't
really have to follow the sequences according to
their exact correlations with the amino acids.
(Slide.)
DR. KASARDA: Now, this is a list of some
of the either toxic or immunoactive peptides that
have been described in the literature. This is
from Sean, et al, from Chaitan Khosla's lab at
Stanford. The 33-Mer appears to be a very active
sequence. I have indicated some
sort of homology
100
here by the yellow boxes here.
All of these sequences, with the exception
of this one, have been found to be toxic by direct
installation into the small intestine or they have
been found to stimulate T-cells, T-cell clones,
derived from biopsies of celiac patients.
These are just some of the toxic
sequences. We don't know all of the toxic
sequences at this point. There are certainly
others to be found, so it is a pretty complicated
situation in trying to sort out exactly what it is
about the sequences that produces toxicity in
celiac disease.
(Slide.)
DR. KASARDA: Now I want to move on and
talk a little bit about the other grains. If we
start with the class flowering plants, which is one
of the major divisions or plants in terms of
taxonomy.
We go down to the major two subclasses,
monocotyledones plants and dicotyledones plants.
101
We can follow the monocots down to the Gramineae or
the grass family.
Here, we have only wheat, rye and barley
that are toxic. Triticale is a cross between wheat
and rye, and so would be expected to be toxic.
Now, oats I have had to put in both
columns, and I will explain why. There are many
other grasses in which the grains do not have toxic
proteins as far as we know.
There are only two grains that have been
studied with modern methods and modern approaches
to understanding their relationship to celiac
disease, and that is wheat and oats.
These others have often been studied very
minimally including rye and barley, but rye and
barley do contain proteins that have sequences
quite close to those in wheat.
We assume that rye and barley are probably
toxic grains according to the early results of
Dicke in the Netherlands back around 1950, where
they considered rye, barley and oats as part of the
toxic
group.
102
Now about 15 years ago, I suggested that
if there are only a few grasses that contain the
toxic sequences, and they are closely related as
you will see, and then there are many other grasses
that do not contain the toxic sequences.
I suggested that if you get into the dicot
group -- the buckwheat, quinoa, amaranth, and these
other grains -- it would not be toxic, simply
because of their distant taxonomic relationship to
wheat.
I was a little bit apprehensive about
suggesting this, but over the past 15 years since I
suggested this, people have been eating these other
grains. As far as I know, there hasn't been any
serious indication that these do, in fact, have
toxicity for celiac patients.
There have been some very fine studies
from Finland and throughout the world, indicating
that oats were safe for celiac patients. But
towards the end of last year the Oslo, Norway,
group under Knut Lundin and Ludvig Sollid. They
have found
-- well, I'm getting a little bit ahead
103
of myself. Let's deal with this slide first.
(Slide.)
DR. KASARDA: If we take a subfamily,
festucoidiae, of the grass family and we look at
the tribal level, and I made this slide before the
results from Oslo were published. The hordeae --
which includes wheat, rye, and barley -- were one
tribe. I thought that oats were probably
non-toxic, so I put them in -- well, they belong in
a separate tribe. It was only this one tribe that
had the toxic sequences.
Now, it is pretty certain that the oats
are toxic to a few probably rare individuals, but
we don't really know how this works out. They
found three celiac patients who definitely reacted
to oats by the same mechanism that they reacted to
gliadin peptides. This, I think, was pretty well
demonstrated by the work from Norway.
Now, here I show the proteins that you
find in wheat, gamma-type gliadins and related
low-molecular-weight glutenin subunits, they are
found in
rye, barley, not in oats. Alpha-type
104
gliadins are found in wheat, but you don't find
them in rye and barley or in oats, and so on down
the line.
Now, the avenins are a small fraction of
the total proteins in oats. These make up only
about 10 percent of the protein. Most of the
protein is oat globulin, which as far as we know if
not harmful or toxic to celiac patients. There are
low-molecular weight proteins related to the
avenins in rye, barley, and wheat.
(Slide.)
DR. KASARDA: Now, this is another one of
those sequence slides, but let me just try to make
a few points here. This top sequence is a gliadin
sequence. It starts here (indicating) and runs
down to here.
The bottom sequence is an avenin, which
shows a lot of homology with the C-terminal half of
this gamma-gliadin molecule. This is also true for
the alpha gliadins. Where the amino acids are the
same, I have colored them in blue.
There is a lot of homology here in the
105
C-terminal half, but that is not where the toxicity
lies. The Oslo group has shown that this
particular sequence, which I have underlined, does